Shamoon et al. BMC Medical Genetics(2020) 21:203
https://doi.org/10.1186/s12881-020-01141-8

RESEARCH ARTICLE

Open Access

Genotype-phenotype correlation of HbH
disease in northern Iraq
Rawand P. Shamoon1,2* , Ahmed K. Yassin3,4, Ranan K. Polus1 and Mohamad D. Ali4

Abstract
Background: HbH disease results from dysfunction of three, less commonly two, α-globin genes through various
combinations of deletion and non-deletion mutations. Characterization of the mutations and the underlying
genotypes is fundamental for proper screening and prevention of thalassaemia in any region. The aim of this study
was to explore the genetic arrangements of HbH disease and to correlate the genotypes with the clinicalphenotypes.
Methods: A total of 44 HbH disease patients were enrolled in this study. They were clinically and haematologicallyassessed. The patients were tested for 21 common α-globin gene mutations using multiplex PCR and reverse
hybridization. According to the genotype, the patients were categorized into two separate sub-groups, deletionand non-deletion types HbH disease.
Results: Within the studied HbH disease patients, eight different α-globin gene mutations were detected in ninedifferent genetic arrangements. The --MED and -α3.7 deletions were the two most frequently encountered mutations(37.5 and 35.2% respectively). Patients with deletion genotypes constituted 70.4%. The most common detected
genotype was --MED/−α3.7 (59.1%), followed by αpoly-A1α/αpoly-A1α (13.6%). For the first time, coinheritance of two
relatively mild mutations (−α3.7/ααAdana) was unpredictably detected in a 1.5 year-old child with Hb of 7.1 g/dL.
Conclusion: The HbH disease patients’ clinical characteristics were variable with no ample difference between thedeletion and non-deletion types. These results can be of benefit for the screening and management of
thalassaemia in this region.
Keywords: HbH disease, Genotype-phenotype correlation, Alpha-thalassaemia, Erbil, Iraq

Backgroundα-thalassaemia is said to be the most common monogenic disorder worldwide. The genetic defects in the αglobin genes, which are mostly gene deletions, result in
the absence or reduced production of α-globin chain in
the hemoglobin tetramer [1]. Numerous mutations, deletion and non-deletion types, have been so far identified
in the α-globin genes; some causes dysfunction of one αglobin gene, denoted as α+ mutations; others resulting in* Correspondence: rawand.shamoon@hmu.edu.krd
1
Department of Pathology, College of Medicine, Hawler Medical University,
Erbil, Iraq
2
Lab. Section, Thalassemia Care Center, Erbil, Iraq
Full list of author information is available at the end of the article

deletion or deactivation of two α-globin genes, denoted
as α0 alleles [2]. The clinical manifestations of these genetic defects vary. In heterozygous state, these mutations
are usually of little clinical significance. However, thecoinheritance of these mutations may result in a variety
of clinical and haematological phenotypes. The most severe phenotype is hydrops fetalis, where all four α-globin
genes are missing (−−/−−). Hemoglobin H (HbH) disease is a moderate clinical form of α-thalassaemia in
which three of the four α-globin genes are affected (−−/−α). The unstable HbH can precipitate in the red cells
causing hemolysis and jaundice [3–5]. Less commonly,
HbH disease may result from two point mutations in the
α-globin genes [6–8]. Based on the nature of the genetic© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Shamoon et al. BMC Medical Genetics(2020) 21:203

defects, two types of HbH disease exist. The first, andcommonest, form is called deletion HbH disease that results from a large deletion mutation, which deactivates
both α-globin genes on one chromosome such as --MED,−-SEA, −-Thai, or --FIL combined with a single-gene deletion
like -α3.7 or -α4.2 on the other chromosome. The second
type is the non-deletion form of HbH disease, which ensues when a non-deletion mutations in either the α1 or α2globin genes on one chromosome combines with anothernon-deletion mutation, or with a deletion of both α-globin
genes on the other chromosome [9, 10].
α-thalassaemia mutations, which are apparently notcommon in this geographical location, have not beenstudied before, nor HbH disease genotypes were explored. This study aimed to characterize the α-globin
gene mutations in this cohort of patients, discover the
mutation combinations resulting in HbH disease, andcorrelate the disease genotypes with clinical and haematological phenotypes.

Methods
Over 6 years, starting from February 2014, 44 cases of
HbH disease were recruited to this study at Erbil Thalassaemia Care Centre, Erbil, Northern Iraq. Most of theenrolled patients were referred to the aforementioned
centre from other hospitals and/or private clinics withthe suspicion of having a hemoglobinopathy. All enrolled
patients were interviewed and physically examined.
Demographic data and disease history, specifically transfusion history, were obtained. Informed written consent
was obtained from the patients. In the case of minors(less than 16 years), the guardian’s consent was obtained
before obtaining their assent. The study was approvedby the Research Ethics Committee of Hawler Medical
University, Erbil, Iraq. The diagnosis of HbH disease wasmade following performing routine complete bloodcount, using automated haematology analyzer (Swelab,
Spagna, Sweden), and peripheral blood smear; HbH inclusion test, using 1% brilliant cresyl blue staining of the
red cells and incubation at 37 °C for 1 h; and measuring
the HbH fraction using high performance liquid chromatography (HPLC), with the VARIANT™ HPLC systemon fresh blood samples (Bio-Rad Laboratories, Hercules,
CA, US) [11]. Serum ferritin level was measured with
Cobas E411 analyzer, Roche®, Germany.
The molecular analysis started with DNA extraction
using Gentra Puregene blood kit (Qiagen, Germantown,
MD, USA). α-globin genotyping was then performed
using multiplex PCR and reverse hybridization assay according to the manufacturer’s instructions (Alpha-globin
StripAssay; ViennaLab Diagnostics, Vienna, Austria), covering the following 21 mutations: two single gene deletions (−α3.7, −α4.2), five double gene deletions [−-MED,−-SEA, −-THAI, −-FIL, −α20.5 kb], the αααanti-3.7 gene

Page 2 of 6

triplication, two point mutations on the α1 gene [codon
14 (TGG > TAG); codon 59 (GGC > GAC) (Hb Adana)],and 11 mutations on the α2 gene [initiation codon ATG >ACG; codon 19 (GCG > GC-), IVS-I(− 5 nt) (−TGAGG);
codon 59 (GGC > GAC); codon 125 (CTG > CCG) (Hb
Quong Sze); Hb Constant Spring (HbCS) codon 142,
Term → Gln, TAA > CAA; Hb Icaria, codon 142, Term→
Lys, TAA > AAA; Hb Pakse´, codon 142, Term→ Tyr,
TAA > TAT, Hb Koya Dora, codon 142, Term→ Ser,TAA > TCA; polyadenylation signal site, poly-A1(AATAAA>AATAAG); and poly-A2 (AATAAA>AATGAA) [12]. Based on the nature of the α-globin genetic defects, and for the sake of comparing patients’ phenotypiccharacteristics, we categorized the patients into two distinct genotypic groups: deletion and non-deletion HbH
disease subgroups. In the latter subgroup, we included patients with both non-deletion/non-deletion and the deletion/non-deletion arrangements together.
Statistical analysis was performed using Mircosoft®
Excel, Professional Edition, 2010. Data was described in
number and percentage. Quantitative data were described using mean, standard deviation median, andrange. Chi-square test was used to compare categorical
data. Mann-Whitney test was used to compare two setsof numerical data. Correlations between two quantitative
variables were assessed using Spearman’s coefficient. Significance was considered at P < 0.05.

Results
Over 6 years, 44 HbH disease patients, 19 males and 25 females, were recruited to this study. Their age ranged from
1 to 68 years, with a median of 19 years. The median Hb
was 9.15 g/dL, ranging from 6.4 to 12.7. Table 1 showssome demographic and clinical parameters of the patients.
Serum ferritin showed a positive correlation with age(r = 0.761) (Fig. 1). Splenectomized patients were 5
Table 1 Demographic and clinical characteristics of patientswith HbH disease
Parameters
Age, y (median, range)

19 (1–68)

GenderMale

19 (43.2%)

Female25 (56.8%)

Hb, g/dL (median, range)

9.15 (6.4–12.7)

HbH, % (median, range)

7.35 (1–25.7)

Ferritin, ng/mL (median, range)

442 (11–2950)

Splenectomized

5 (11.4%)

Transfusion status
Never transfused

23 (52.3%)

Occasional (≤1 unit per year)

17 (38.6%)

Irregular (2 to 4 units per year)

4 (9.1%)

Shamoon et al. BMC Medical Genetics(2020) 21:203

Page 3 of 6

(13.6%) patients. Deletion genotypes we encountered in
31 patients (70.45%), while non-deletion genotypes wereobserved in 13 cases (29.54%). The α-globin genotypes
and their relative frequencies are illustrated in Table 3.
Some relevant clinical and haematological parameters
within the deletion and non-deletion HbH disease subgroups are illustrated in Table 4. The median values of
the red cell count and Hb were higher in patients withdeletion HbH disease; however, the differences were notstatistically significant. All other haematological andclinical parameters did not show significant differences
between the two groups.
Fig. 1 Correlation of serum ferritin with the age of HbH
disease patients(11.4%), we found their mean age was noticeably higher
compared to the non-splenectomized (34.6 versus 20.5
years respectively, p = 0.05). A significant relation (p <0.001) was found between patients’ age and blood transfusion. There were 23 (52.3%) patients who never received a transfusion; their mean age was 10.5 years
versus 35.4 years in the transfused patients. The Hb andferritin levels were not different in the splenectomized
and non-splenectomized patients (p-values 0.061 and
0.138, respectively). We also found significantly higher
ferritin levels among transfused patients (p < 0.001).
The molecular characterization detected eight different
α-globin mutations within the studied group. As illustrated in Table 2, −-MED double-gene deletion was themost common mutation, which was observed in 33(37.5%) alleles, followed by mutations of -α3.7 (35.2%),α2poly-A1 (15.9%), −α4.2 (3.4%), in addition to four othermutations which collectively comprised 8% of all mutatedalleles. Totally, deletion mutations constituted 78.4% andnon-deletion mutations 21.6% of all mutated alleles.
The mutated alleles were found in nine different genetic arrangements. The most frequent genotype was--MED/−α3.7 arrangement, which was detected in 26(59.1%) patients. The second most common genotype
was homozygosity of αPoly-A1α, which was observed in 6

Discussion
HbH disease is considered the most symptomatic formof α-thalassaemias that is compatible with life. The disease, however, has a heterogeneous clinical and haematological phenotype ranging from very mild phenotypes
to more severe forms requiring regular or irregularblood transfusion. The severity of the disease is assumed
to be related to the degree of α:β globin chain imbalance,
which is principally determined by the nature of theunderlying α-globin mutations [1, 10]. However, variations in the clinical phenotypes have been observed evenin the presence of similar genotype, which is probably
due to genetic and environmental modifiers [13].
Erbil province in northern Iraq covers an area of 15,
075 km2 with a population of about two million people.
Majority of the population are Kurds; the minorities of
Turkmen, Assyrians, and Arabs comprise about 5% of its
total population. The province is bordered by Turkey
from the north-east and Iran from the east. We knowmuch about β-thalassaemia in northern Iraq. Many studies have described the spectrum of β-globin gene mutations, the carrier rate, and the genotypic mechanisms inboth thalassaemia major and thalassaemia intermedia[14–19]. Scrutinizing α-thalassaemia is not as easy as for
β-thalassaemia because of the lower carrier rate in our
region, and the difficulty in confirming diagnosis of αthalassaemia carriers via the routine Hb-electrophoresis.

Table 2 Mutation frequency in HbH disease patients
MutationType

Frequency

%

--MED

Deletion

33

37.5

-α

3.7

Deletion31

35.2

α2poly-A1 (AATAAA>AATAAG)

Point mutation

14

15.9

-α4.2

Deletion33.4-α20.5

Deletion2

2.3

Codon 59 (GGC > GAC) (Hb Adana)

Point mutation

2

2.3

α2 IVS-I(−5 nt) (−TGAGG)

Splicing site 5 nt. Deletion22.3

α2poly-A2 (AATAAA>AATGAA)

Point mutation

Total

1

1.1

88

100

Shamoon et al. BMC Medical Genetics(2020) 21:203

Page 4 of 6

Table 3 HbH disease genotypes
Genotypes

Frequency%--MED/−α3.7

26

59.1

Αpoly-A1α/αpoly-A1α

6

13.6

4.2

3

6.8

-α20.5/−α3.7

2

4.5

MED/

--

MED

--

−α

ivs1(−5 nt)

/α

α

2

4.5

-α3.7/αpoly-A1α

2

4.5

α

1

2.3

--MED/ααAdana

1

2.3

MED

--

poly-A2/α

3.7-α /αα

Adana

Total

1

2.3

44

100

The carrier rate of α-thalassaemia in Iraq is estimated at< 1%, but no study has yet rectified the exact carrier ratein this region. So far, a couple of small studies by AlAllawi et al. have described the spectrum of α-globin
gene mutations among carriers in the neighboring provinces of Dohuk and Sulaimaniyah [20, 21]. Results of thecurrent study, which is the first to tackle a relatively
large cohort of patients with HbH disease in this region,
will be complementary to improve the existing national
thalassemia carrier screening and genetic counselingprogram that was established in 2009, and will be helpful
for prenatal diagnosis.
The clinical and haematological features of the enrolled patients showed obvious heterogeneity. Substantial variation was noticed in the patients’ age, Hblevel, and transfusion requirement. Many clinical parameters strongly related to patients’ age, primarily
the ferritin level and transfusion requirement. It is
widely reported that the clinical severity of thalassaemias and other hemoglobinopathies is extremely age
dependent [22].

In the current study, we have identified eight different
α-globin gene mutations, four of which are deletion mutations (−-MED, −α3.7, −α4.2, and -α20.5) and theremaining four are non-deletion mutations (α2 poly-A1,
Hb Adana, α2 IVS1 (− 5 nt), and α2 poly-A2). Comparable to the results of earlier reports by Al-Allawi et al.
[20, 21], we found the spectrum of α-globin gene defects
restricted to a relatively limited number of mutations.
However, this finding is unlike to what have been observed in Iran, Turkey, Sardinia, and Cyprus, where themutations are more heterogeneous [23, 24]. Within our
HbH disease patients, the --MED double-gene deletion
was the most commonly encountered mutation (33 alleles) followed by the worldwide prevalent -α3.7 single
gene deletion (31 alleles). Similar findings have been reported by the vast majority of studies done in the Mediterranean and Middle East regions [25]. The α2 poly-A1
non-deletion mutation, which is known to be prevalent
in the Arabian Peninsula, was encountered in 14 alleles
and was the most frequently encountered non-deletion
mutation. Homozygosity for the latter mutation is themain genotype causing HbH disease in Saudi Arabia,
UAE, Kuwait, and Bahrain [26–29]. None of the southeast Asian mutations were observed in this cohort. A
previous study has reported the --FIL double-gene deletion in one β-thalassaemia intermedia case who washomozygous for IVS-II-1 (G > A). The same deletion
was also observed in Hatay Province of Turkey [18, 25].
We, in the current cohort, observed nine different
genotypes causing HbH disease, most of which weredeletion genotypes (70.45%, 31 patients). The --MED/−α3.7 arrangement was the most frequently encountered genotype as in the neighboring areas of Turkey
and Iran. High prevalence of deletion type HbH disease have been reported in the Mediterranean area,
Turkey, Iran, Greece, and Sardinia, (80, 50.5, 56.1,

Table 4 Clinical and haematological parameters in the deletion and non-deletion types HbH Disease
p-valueParameters

Deletion(31 cases)

Non-Deletion(13 cases)

RBC count, (median, range)

5.05 (3.5–6.61)

4.51 (2.96–6.74)

0.069

Hb, g/dL (median, range)

9.2 (8.0–11.5)

8.3 (6.4–12.7)

0.086

MCV, fl (median, range)

61.3 (52.2–68.7)

62.6 (52.5–81.9)

0.309

MCH, pg (median, range)

18.0 (16.1–21.9)

18.9 (16.1–28)

0.241

RDW, % (median, range)

25.6 (16.9–30.7)

25.0 (18.7–36.3)

0.816

HbH, % (median, range)

7.5 (1–15)

7.2 (5.7–25.7)

0.125

Ferritin, ng/mL (median, range)

467 (11–2950)

389 (29–1726)

0.403

Splenectomized

4 (12.9%)

1 (7.7)0.20

Never transfused

16 (51.6%)

7 (53.8%)

0.57

Occasional (≤1 unit per year)

13 (41.9%)

4 (30.8%)

Irregular (2 to 4 units per year)

2 (6.5%)

2 (15.4%)

Transfusion

Shamoon et al. BMC Medical Genetics(2020) 21:203

and 86% respectively) [10, 26, 30, 31]. We detected
19 alleles carrying non-deletion mutations of which
17 involved the α2 gene. Mutations of the α2-globin
gene are associated with more severe phenotype because more than two-third of the α-globin chain synthesis is controlled by the dominant α2 globin genetranscription [32]. Hb Adana was the only detected
α1 globin gene mutation in our patients; it was observed in compound heterozygosity with --MED deletion in one patient and with -α3.7 deletion in another.
We could not find any previous study reporting thelatter combination (−α3.7/ααAdana) to have a HbH
equivalent phenotype; though, we surprisingly foundit in a 1.5 year-old boy with Hb of 7.1 g/dL. Coinheritance of -α3.7 deletion, with many other point mutations in the α2 globin gene, are reported in manystudies [33]. In our cohort, coinheritance of two point
mutations was detected in six patients (13.6%) only,
who all were homozygous for α2 poly-A1 mutation.
Non-deletion genotypes are reported to be the predominant HbH disease genotypes in the Arabia area,
Jordan, and northern Iran [23, 27, 34, 35].
It has been previously documented that patients withdeletion/deletion HbH disease genotype are of least severe phenotype, followed by deletion/non-deletion arrangement, and the non-deletion/non-deletion genotype
which is supposed to be the most severe [6, 30]. In ourcohort, we did not find any significant difference in theclinical and haematological parameters, which we studied in both deletion and non-deletion HbH disease subgroups. The median Hb and red cell count were notably
lower in the non-deletion HbH subgroup, however not
to a statistically significant level. The remaining red cellparameters, the ferritin level, the HbH fraction, splenectomy, and blood transfusion status did not show any difference in the two subgroups.

Conclusion
This study showed that our HbH disease patients hadvariable clinical and haematological phenotypes ranging
from very mild cases not requiring transfusion to more
severe others needed irregular transfusions. The
spectrum of α-globin gene mutations and then theunderlying genotypic arrangements were not many. The
majority of our HbH disease patients were found to havedeletion genotypes; however, their clinical phenotypes
did not differ much from those having non-deletion genotypes. Knowing all these will definitely have a positive
impact on thalassaemia diagnosis and management
guidelines in this region.
Abbreviations
HbH: Hemoglobin-H; PCR: Polymerase chain reaction; HPLC: Highperformance liquid chromatography; Hb: Hemoglobin; RBCs: Red blood cells;

Page 5 of 6

MCV: Mean cell volume; MCH: Mean cell hemoglobin; RDW: Red cell
distribution width
Acknowledgements
The authors very much appreciate the assistance and support provided by
Erbil Thalassemia Care Centre and the Lab Division of PAR Hospital.
Authors’ contributions
RPS contributed in conceiving and designing the study, molecular testing,analyzing the data, and editing and finalizing the manuscript. AKY and MDA
contributed in conceptualization and designing the study and took part inwriting the initial draft of the manuscript. RKP contributed inconceptualization and designing the study, data collection, and the initial
draft of the manuscript. The author(s) read and approved the final
manuscript.
Funding
Not applicable.
Availability of data and materials
The dataset used for the current study is available in the Mendeley datasets
repository (DOI: https://doi.org/10.17632/bjgf3kcpgk.1). https://data.mendeley.
com/datasets/bjgf3kcpgk/1
Ethics approval and consent to participate
The study protocol was reviewed and approved by the Research Ethics
Committee at Hawler Medical University. Each participant provided written
informed consent before participation in the study. In the case of minors,
the guardian’s written consent was obtained before obtaining their assent.
The anonymity of participants and the confidentiality of collected
information were explained for the participants.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Pathology, College of Medicine, Hawler Medical University,
Erbil, Iraq. 2Lab. Section, Thalassemia Care Center, Erbil, Iraq. 3Department ofInternal Medicine, College of Medicine, Hawler Medical University, Erbil, Iraq.
4
Department of Clinical Hematology, Nanakali Hemato-Oncology Teaching
Center, Erbil, Iraq.
Received: 9 April 2020 Accepted: 8 October 2020

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